Exposure of cells to insulin promotes phosphorylation of the insulin receptor's cytoplasmic domain on serine, threonine and tyrosine. Insulin- dependent tyrosine phosphorylation probably reflects receptor autophosphorylation, and the receptor is known to be only a tyrosine kinase. Thus, the serine and threonine phosphorylation observed most likely reflects receptor-induced phosphorylation by an exogenous kinase(s). Serine phosphorylation of the insulin receptor has been correlated with decreases, in the receptor's intrinsic tyrosine kinase and with decreases in insulin's ability to stimulate cellular metabolism. Therefore, serine phosphorylation of the insulin receptor may be an important regulator od insulin action. We have discovered an affinity-purified insulin receptor preparation which contains a copurifying serine/threonine kinase phosphorylates the insulin receptor on several sites phosphorylated in the intact cell identification of these serine and threonine phosphorylation sites makes possible experiments to explore the role of these phosphorylated residues in controlling receptor serine and threonine phosphorylation sites--via expression of mutant receptors lacking specific serines and threonines--on insulin signalling in the intact cell. The following questions will be addressed: 1. Does site-directed mutagenesis of serine and threonine phosphorylation sites change the pattern and level of insulin receptor tyrosine kinase autophosphorylation in intact cells? Comparison of phosphopeptide maps from normal insulin receptors and insulin receptors mutated at serine or threonine phosphorylation sites may reveal the point(s) in the receptor autophosphorylation cascade disrupted by these phosphorylations. 2. What effect does the alteration of serine of threonine phosphorylation sites by site-directed mutagenesis have on the rate of receptor internalization and insulin-dependent receptor down-regulation? Recent reports indicate that mutant insulin receptors lacking tyrosine kinase activity are not phosphorylated on serine or threonine, nor can these receptors undergo ligand-induced down-regulation. 3. Does the alteration of one or more sites of serine/threonine phosphorylation by site-directed mutagenesis modify insulin signalling? Serine phosphorylation of the insulin receptor correlates well with decreased insulin activation of glycogen synthase and tyrosine aminotransferase. If insulin receptor serine/threonine phosphorylation is an important regulator of insulin action the loss of these phosphorylation sites may enhance and prolong cellular responses to insulin. Detailed investigation of these questions will provide fundamental information regarding the biochemical mechanisms that control insulin signalling and may provide a useful model for exploring insulin resistance at the cellular level.